Serum Cytokine and Growth Factor Levels in Children with Autism Spectrum Disorder.

BACKGROUND The immune system may have a role in the pathogenesis of autism spectrum disorder (ASD), including typical and atypical autism. The aim of this study was to determine whether a cytokine and growth factor panel could be identified for the diagnosis and prognosis in children with ASD, including typical and atypical autism. MATERIAL AND METHODS This study included 26 children with ASD (typical or atypical) and 11 of their siblings who did not have ASD. A panel of ten serum cytokines and growth factors were investigated using addressable laser bead assay (ALBIA) and enzyme-linked immunosorbent assay (ELISA) kits. Results were correlated with scores using the Childhood Autism Rating Scale (CARS) and Autism Diagnostic Observation Schedule (ADOS) for the children with ASD and compared with the findings from their siblings without ASD. RESULTS There were no statistically significant differences in serum cytokine and growth factor levels between children with ASD and their siblings. The scores using CARS and ADOS were significantly greater in children with typical autism compared with children with atypical autism as part of the ASD spectrum. Serum levels of cytokines and growth factors showed a positive correlation with CARS and ADOS scores but differed between children with typical and atypical autism and their siblings. CONCLUSIONS The findings of this study showed that serum measurement of appropriately selected panels of cytokines and growth factors might have a role in the diagnosis of ASD.

Genotoxicity of 7H-dibenzo[c,g]carbazole and its methyl derivatives in human keratinocytes.

Differences between tissues in the expression of drug-metabolizing enzymes may substantially contribute to tissue-specificity of chemical carcinogens. To verify this hypothesis, the spontaneously immortalized human keratinocytes HaCaT were used, in order to evaluate the genotoxic potential of 7H-dibenzo[c,g]carbazole (DBC), a known hepatocarcinogen and sarcomagen, and its synthetic tissue-specific derivatives, 5,9-dimethyl-DBC (DiMeDBC) and N-methyl-DBC (N-MeDBC), which manifest specific tropism to the liver and skin, respectively. HaCaT cells mainly express cytochrome P4501A1 (CYP1A1), which is involved in metabolism of DBC and N-MeDBC, but not DiMeDBC [10]. Both DBC and the sarcomagen N-MeDBC induced significant levels of DNA strand-breaks, micronuclei, and DNA adducts followed by the phosphorylation of the p53 protein and histone H2AX in HaCaT cells. In contrast, the specific hepatocarcinogen DiMeDBC was devoid of any significant genotoxic activity in this cell line. Our study demonstrates that the absence of drug-metabolizing enzyme(s) involved in DiMeDBC metabolism may contribute substantially to the tissue-specific genotoxicity of this hepatocarcinogen.

Toxic effects of methylated benzo[a]pyrenes in rat liver stem-like cells.

The methylated benzo[a]pyrenes (MeBaPs) are present at significant levels in the environment, especially in the sediments contaminated by petrogenic PAHs. However, the existing data on their toxic effects in vitro and/or in vivo are still largely incomplete. Transcription factor AhR plays a key role in the metabolic activation of PAHs to genotoxic metabolites, but the AhR activation may also contribute to the tumor promoting effects of PAHs. In this study, the AhR-mediated activity of five selected MeBaP isomers was estimated in the DR-CALUX reporter gene assay performed in rat hepatoma cells. Detection of other effects, including induction of CYP1A1, CYP1B1, and AKR1C9 mRNAs, DNA adduct formation, production of reactive oxygen species, oxidation of deoxyguanosine, and cell cycle modulation and apoptosis, was performed in the rat liver epithelial WB-F344 cell line, a model of liver progenitor cells. We identified 1-MeBaP as the most potent inducer of AhR activation, stable DNA adduct formation, checkpoint kinase 1 and p53 phosphorylation, and apoptosis. These effects suggest that 1-MeBaP is a potent genotoxin eliciting a typical sequence of events ascribed to carcinogenic PAHs: induction of CYP1 enzymes, formation of high levels of DNA adducts, activation of DNA damage responses (including p53 phosphorylation), and cell death. In contrast, 10-MeBaP, representing BaP isomers substituted with the methyl group in the angular ring, elicited only low levels DNA adduct formation and apoptosis. Other MeBaPs under study also elicited strong apoptotic responses associated with DNA adduct formation as the prevalent mode of toxic action of these compounds in liver cells. MeBaPs induced a weak production of ROS, which did not lead to significant oxidative DNA damage. Importantly, 1-MeBaP and 3-MeBaP were found to be potent AhR agonists, one order of magnitude more potent than BaP, thus suggesting that the AhR-dependent modulations of gene expression, deregulation of cell survival mechanisms, and further nongenotoxic effects associated with AhR activation may further contribute to their tumor promotion and carcinogenicity.

Differences in DNA damage and repair produced by systemic, hepatocarcinogenic and sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitor cells.

Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens. Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9-dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole (N-MeDBC), induced several cellular events associated with tumor promotion in WB-F344 cells, an in vitro model of liver oval cells [J. Vondracek, L. Svihalkova-Sindlerova, K. Pencikova, P. Krcmar, Z. Andrysik, K. Chramostova, S. Marvanova, Z. Valovicova, A. Kozubik, A. Gabelova, M. Machala, 7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial 'stem-like' cells, Mutat. Res. Fundam. Mol. Mech. Mutagen. 596 (2006) 43-56]. In this study, we focused on the genotoxic effects generated by these dibenzocarbazoles in WB-F344 cells to better understand the cellular and molecular mechanisms involved in hepatocarcinogenesis. Lower IC(50) values determined for DBC and DiMeDBC, as compared with N-MeDBC, indicated a higher sensitivity of WB-F344 cells towards hepatocarcinogens. Accordingly, DBC produced a dose-dependent DNA-adduct formation resulting in substantial inhibition of DNA replication and transcription. In contrast, DNA-adduct number detected in DiMeDBC-exposed cells was almost negligible, whereas N-MeDBC produced a low level of DNA adducts. Although all dibenzocarbazoles significantly increased the level of strand breaks (p<0.05) and micronuclei (p<0.001) after 2-h treatment, differences in the kinetics of strand break rejoining were found. The strand break level in DiMeDBC- and N-MeDBC-exposed cells returned to near the background level within 24h after treatment, whereas a relatively high DNA damage level was detected in DBC-treated cells up to 48h after exposure. Additional breaks detected after incubation of DiMeDBC-exposed WB-F344 cells with a repair-specific endonuclease, along with a nearly 3-fold higher level of reactive oxygen species found in these cells as compared with control, suggest a possible role of oxidative stress in DiMeDBC genotoxicity. We demonstrated qualitative differences in the DNA damage profiles produced by hepatocarcinogens DBC and DiMeDBC in WB-F344 cells. Different lesions may trigger distinct cellular pathways involved in hepatocarcinogenesis. The low amount of DNA damage, together with an efficient repair, may explain the lack of hepatocarcinogenicity of N-MeDBC.

The aryl hydrocarbon receptor-mediated and genotoxic effects of fractionated extract of standard reference diesel exhaust particle material in pulmonary, liver and prostate cells.

Diesel exhaust particles (DEP) and the associated complex mixtures of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), or their derivatives, have been suggested to exert deleterious effects on human health. We used a set of defined cellular models representing liver, lung and prostate tissues, in order to compare non-genotoxic and genotoxic effects of crude and fractionated extract of a standard reference DEP material - SRM 1650b. We focused on the aryl hydrocarbon receptor (AhR)-mediated activity, modulation of cell proliferation, formation of DNA adducts, oxidative DNA damage, and induction of DNA damage responses, including evaluation of apoptosis, and phosphorylation of p53 tumor suppressor and checkpoint kinases (Chk). Both PAHs and the polar aromatic compounds contributed to the AhR-mediated activity of DEP-associated organic pollutants. The principal identified AhR agonists included benzo[k]fluoranthene, indeno[1,2,3-c,d]pyrene, chrysene and several non-priority PAHs, including benzochrysenes and methylated PAHs. In contrast to PAHs, polar compounds contributed more significantly to overall formation of DNA adducts associated with phosphorylation of p53, Chk1 or Chk2, and partly with apoptosis. Therefore, more attention should be paid to identification of DEP-associated polar organic compounds, contributing to the AhR activation and cytotoxic/genotoxic effects of complex airborne mixtures of organic contaminants produced by diesel engines.

Genotoxicity of 7H-dibenzo[c,g]carbazole and its tissue-specific derivatives in human hepatoma HepG2 cells is related to CYP1A1/1A2 expression.

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression.

Genotoxic polycyclic aromatic hydrocarbons fail to induce the p53-dependent DNA damage response, apoptosis or cell-cycle arrest in human prostate carcinoma LNCaP cells.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been positively associated with prostate cancer, but knowledge of the formation of PAH-DNA adducts and related genotoxic events in prostatic cells is limited. In the present study, benzo[a]pyrene (BaP), a potent mutagenic PAH, formed significant levels of DNA adducts in cell lines derived from human prostate carcinoma. When analyzing the effect of BaP on the induction of CYP1 enzymes participating in the metabolic activation of PAHs in LNCaP cells, we found that BaP induced expression of CYP1A1 and CYP1A2, but not CYP1B1 enzyme. Despite a significant amount of DNA adducts being formed by BaP and, to a lesser extent also by another strong genotoxin, dibenzo[a,l]pyrene, neither apoptosis nor cell-cycle arrest were induced in LNCaP cells. LNCaP cells were not sensitized to the induction of apoptosis by PAHs even through inhibition of the phosphoinositide-3-kinase/Akt pro-survival pathway. The lack of apoptosis was not due a disruption of expression of pro-apoptotic and pro-survival members of the Bcl-2 family of apoptosis regulators. In contrast to other genotoxic stimuli, genotoxic PAHs failed to induce DNA double-strand breaks, as illustrated by the lack of phosphorylation of histone H2AX or checkpoint kinase-2. BaP did not activate p53, as evidenced by the lack of p53 accumulation, phosphorylation at Ser15, or induction of p53 transcriptional targets. Taken together, although genotoxic PAHs produced significant levels of DNA adducts in a model of human prostate carcinoma cells, they did not activate the mechanisms leading to elimination of cells with significant damage to DNA, presumably due to their failure to activate the p53-dependent DNA damage response.

The role of aryl hydrocarbon receptor in regulation of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons in a model of rat liver progenitor cells.

In contrast to hepatocytes, there is only limited information about the expression and activities of enzymes participating in metabolic activation of environmental mutagens, including polycyclic aromatic hydrocarbons (PAHs), in liver progenitor cells. In rat liver "stem-like" WB-F344 cell line, sharing many characteristics with rat liver progenitor cells, PAHs are efficiently activated to their ultimate genotoxic metabolites forming DNA adducts. The present study aimed to characterize expression/activities of enzymes of two major pathways involved in the metabolism of benzo[a]pyrene (BaP): cytochrome P450 (CYP) family 1 enzymes and cytosolic aldo-keto reductases (AKRs). We report here that, apart from induction of CYP1A1 and CYP1B1 expression and the corresponding enzymatic activity, both BaP and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced rat 3alpha-hydroxysteroid dehydrogenase (AKR1C9) expression and activity. In contrast, the aldehyde reductase AKR1A1 was not induced by either treatment. Thus, both CYP1 and AKR metabolic pathways were inducible in the model of liver progenitor cells. BaP and TCDD were efficient inducers of NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in WB-F344 cells, a principal enzyme of cellular antioxidant defense. Both compounds also induced expression of transcription factor NRF2, involved in control of enzymes protecting cells from oxidative stress. However, although BaP induced a significant formation of reactive oxygen species, it did not induce expression of heme oxygenase-1, suggesting that induction of oxidative stress by BaP was limited. Using shRNA against the aryl hydrocarbon receptor (AhR), we found that similar to CYP1A1 and CYP1B1, the AKR1C9 induction was AhR-dependent. Moreover, constitutive AKR1C9 levels in AhR-deficient rat BP8 hepatoma cells were significantly lower than in their AhR-positive 5L variant, thus supporting possible role of AhR in regulation of AKR1C9 expression. Taken together, both CYP1 and AKR1C9 appear to be AhR-regulated metabolic pathways, which may contribute to formation of pro-carcinogenic PAH metabolites in liver progenitor cells.

Toxic effects of methylated benz[a]anthracenes in liver cells.

Monomethylated benz[ a]anthracenes (MeBaAs) are an important group of methylated derivatives of polycyclic aromatic hydrocarbons (PAHs). Although the methyl substitution reportedly affects their mutagenicity and tumor-initiating activity, little is known about the impact of methylation on the effects associated with activation of the aryl hydrocarbon receptor (AhR)-dependent gene expression and/or toxic events associated with tumor promotion. In the present study, we studied the effects of a series of MeBaAs on the above-mentioned end points in rat liver cell lines and compared them with the effects of benz[ a]anthracene (BaA) and the potent carcinogen 7,12-dimethylbenz[ a]anthracene (DMBA). Methyl substitution enhanced the AhR-mediated activity of BaA derivatives determined in a reporter gene assay, as the induction equivalency factors (IEFs) of all MeBaAs were higher than that of BaA. IEFs of 6-MeBaA and 9-MeBaA, two of the most potent MeBaAs, were more than two orders of magnitude higher than the IEF of BaA. Correspondingly, all MeBaAs induced higher levels of cytochrome P450 1A1 mRNA. Both BaA and MeBaAs had similar effects on the expression of cytochrome P450 1B1 or aldo-keto reductase 1C9 in rat liver epithelial WB-F344 cells. In contrast to genotoxic DMBA, MeBaAs induced low DNA adduct formation. Only 10-MeBaA induced apoptosis and accumulation of phosphorylated p53, which could be associated with the induction of oxidative stress, similar to DMBA. With the exception of 10-MeBaA, all MeBaAs induced cell proliferation in contact-inhibited WB-F344 cells, which corresponded with their ability to activate AhR. 1-, 2-, 8-, 10-, 11-, and 12-MeBaA inhibited gap junctional intercellular communication (GJIC) in WB-F344 cells. This mode of action, like disruption of cell proliferation control, might contribute to tumor promotion. Taken together, these data showed that the methyl substitution significantly influences those effects of MeBaAs associated with AhR activation or GJIC inhibition.